The goal of this project is to test novel therapeutic strategies to combat accelerated graft (Tx) rejection and to dissect cellular/humoral cascade contributing to this fulminant immune response in sensitized hosts. The study uses agents targeting selectively CD4 and/or CD25 (IL-2R) cell surface molecules + low doses of CsA in the treatment of presensitized rat recipients of heterotopic cardiac Tx. The specific aims are: (1) To determine the effectiveness of CD4/CD25 targeted therapies in preventing/treating accelerated Tx loss: Dose response and time course studies for panels of CD4/CD25 mAbs distinct epitope/isotype specificities will be conducted to erase sensitization and abrogate otherwise 24-36h accelerated Tx rejection. CD4 (BWH-4) and CD25 (ART-18) mAbs proved highly effective in pilot experiments, stressing the importance of these new potential targets for immunosuppression in sensitized recipients. (2) To devise principles of optimal mAb selection for therapeutic use: First, radioactive tracer studies with CD4/CD25 mAbs will be performed in sensitized rats and biodistribution patterns correlated with their in vivo efficacy. Second, mAb mediated elimination vs. modulation/inhibition of target cell function will be addressed by comparing the in vivo effects of intact mAb and its F(ab')2 fragments, and analyzing cell phenotype in lymphoid tissues and at the Tx by FMF and immunohistology. (3) To evaluate the effects of applied therapies upon host cell mediated immunity: The in vitro (LMC, MLR, FMF, immunohistology) and in vivo (adoptive transfer) assays will be used to unravel putative feedbacks between mAb(s) and host cell repertoire. Sparing of cells of T suppressor circuit will be studied in parallel with screening for cell alloaggressive functions; the effects of individual cell subsets will be contrasted. (4) To explore the cytokine network in unmodified/mAb conditioned recipients: Two approaches will be undertaken to delineate the contribution of cytokines to accelerated rejection. First, intra-Tx production of cytokines will be assessed by immunohistology. Second, cytokine transcriptional events will be studied in RNA extracted from the Tx/spleen by Northern blot analysis, hybridization with cDNA probes and amplification by PCR. (5) To assess the effects of mAb treatment upon host humoral immunity: The kinetics of anti-donor Ig responses in untreated/mAb treated recipients will be proved by serial screening of sera by FMF, CDC, ADCC and in adoptive transfer studies. Intra-Tx elaboration of IgG, IgM, C3 will be followed by immunohistology. Anti-Ig, anti-idiotype (Id) and anti-Id-Id responses in mAb treated hosts will be evaluated by ELISA techniques.